TAT-乙肝病毒靶向核糖核酸酶融合蛋白原核载体的构建及表达

Construction and expression of prokaryotic expression vector for pTAT-HBV targeted ribonuclease fusion protein

目的:构建蛋白质转导结构域(protein transduction domain,PTD)TAT和乙肝病毒靶向核糖核酸酶(targeted ribonuclease,TR)融合蛋白的原核表达载体,并在大肠杆菌中表达。方法:将已构建的乙肝病毒靶向核糖核酸酶基因、乙肝病毒核心蛋白(HBV core protein,HBVc)基因、人嗜酸性粒细胞来源的神经毒素(human eosinophil derived neurotoxin,hEDN)基因,克隆人含PTD TAT的pTAT原核表达载体,在大肠杆菌BL21(DE3)LysS内,以IPTG诱导融合蛋白表达。表达产物用SDS-PAGE及Western blot分析鉴定。结果:成功地构建了乙肝病毒靶向核糖核酸酶及其两个对照的融合蛋白原核表达载体,并在IPTG诱导下获得特异性表达。结论:Tat-乙肝病毒 靶向核糖核酸酶融合蛋白的构建,为体内应用靶向核酸酶治 疗乙型肝炎奠定了基础。

AIM: To construct Tat-HBV targeted ribonuclease fusion protein prokaryotic expression vector and express it in E. coli. METHODS: The cDNAs encoding HBV targeted ribonuclease, human eosinophil-derived neurotoxin and HBV core protein were respectivly cloned into prokaryotic expression vector pTAT-HA. Recombinant plasmids were transformed into E. coli BL21 ( DE3) LysS, then the transformed cells were induced with IPTG. The expression of the fusion proteins were analyzed by SDS-PAGE and Western blot. RESULTS: The three recombinant plasmids were constructed and expressed after IPTG induction successfully. CONCLUSION: The obtained Tat-HBV targeted ribonuclease fusion protein has laid the foundation for using TR in therapy of HBV infection.